Viruses 2013, 5, 3192-3212; doi:10.3390/v5123192
OPEN ACCESS
viruses
ISSN 1999-4915
www.mdpi.com/journal/viruses
Review
Make Yourself at Home: Viral Hijacking of the PI3K/Akt
Signaling Pathway
Nora Diehl and Heiner Schaal *
Universitätsklinikum Düsseldorf, Institut für Virologie, Universitätsstraße 1, Düsseldorf 40225,
Germany; E-Mail: [email protected]
* Author to whom correspondence should be addressed; E-Mail: [email protected];
Tel.: +49-211-81-12393; Fax: +49-211-81-10856.
Received: 29 October 2013; in revised form: 3 December 2013 / Accepted: 5 December 2013/
Published: 16 December 2013
Abstract: As viruses do not possess genes encoding for proteins required for translation,
energy metabolism or membrane biosynthesis, they are classified as obligatory intracellular
parasites that depend on a host cell to replicate. This genome limitation forces them to gain
control over cellular processes to ensure their successful propagation. A diverse spectrum
of virally encoded proteins tackling a broad spectrum of cellular pathways during most
steps of the viral life cycle ranging from the host cell entry to viral protein translation has
evolved. Since the host cell PI3K/Akt signaling pathway plays a critical regulatory role in
many cellular processes including RNA processing, translation, autophagy and apoptosis,
many viruses, in widely varying ways, target it. This review focuses on a number of
remarkable examples of viral strategies, which exploit the PI3K/Akt signaling pathway for
effective viral replication.
Keywords: PI3K/Akt signaling; viral entry; alternative splicing; mTOR; apoptosis
1. Introduction
The PI3K/Akt signaling pathway controls numerous cellular processes such as glucose metabolism,
protein synthesis, and proliferation. Whereas the basal activity ensures cell survival, inactivation of
PI3K/Akt signaling results in apoptosis. For an infected organism, apoptosis represents an effective
antiviral instrument which is simple but highly effective. Thus, in order to secure its own replication,
the virus must prevent or delay apoptosis. This may be achieved by maintaining the basal stimulatory
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activity of the PI3K/Akt signaling pathway. Since disabling host cell apoptosis is likely to be an
obligatory step in the viral life cycle [1], regulation of the PI3K/Akt signaling pathway by viruses is
quite likely to have had a considerable evolutionary impact. In response to this viral hijacking of key
cellular processes, the PI3K/Akt pathway appears to be involved in the host cell immune response as a
form of “adaptive strategy” to counteract viral invasion. Thus, when apoptosis is blocked by the virus,
the PI3K/Akt signaling pathway induces expression of interferon-responsive genes [2–6]. Nevertheless,
as a large number of viruses rely on PI3K activity for their replication, there would appear to be a
greater benefit for the virus in activating rather than suppressing the PI3K/Akt signaling pathway.
PI3K/Akt Signaling
The family of phosphoinositide 3-kinases (PI3Ks) is divided into three classes, based on
(i) subcellular distribution; (ii) activating signals; and (iii) substrate specificity (for review see [7]).
The best studied and presumably most relevant class when concerning viral targeting is the class I of
PI3Ks, the members of which are composed of a regulatory (p85) and a catalytic subunit (p110) (for
review see [8]).
When growth factors or cytokines are sensed by their receptor tyrosine kinases (RTKs) or
G protein-coupled receptors (GPCRs), PI3K phosphorylates the 3-hydroxyl group of the inositol ring
of membrane bound phosphatidylinositol (PtdIns) lipid substrates, generating phosphatidylinositol3,4,5-trisphosphates (PIP3), which subsequently serve as docking stations for proteins that harbor lipid
binding domains [8] (Figure 1). The most prominent effector of PI3K is the serine/threonine kinase
Akt (for review see [9]). After binding of its pleckstrin homology (PH) domain to PIP3, Akt
becomes phosphorylated at Thr308 by the phosphoinositide-dependent kinase 1 (PDK1) and at Ser473
by mammalian target of rapamycin complex 2 (mTORC2) leading to its full activation. Notably,
stimulus-dependent, as in the case of the DNA-damage response, Ser473 is phosphorylated
by the DNA-dependent protein kinase (DNA-PK) [10,11]. Finally, several downstream targets like
tuberous sclerosis protein 2 (TSC2; translation), glycogen synthase kinase 3 (GSK3; cell growth), or
Bcl-2-associated death promoter (BAD; cell survival) and forkhead box protein (FOXO; cell survival)
are altered in their function through the Akt-mediated phosphorylation. Termination of this signaling
cascade can occur through the dephosphorylation of PIP3 either by the phosphatase and tensin
homolog (PTEN) or the Src homology domain 2 containing inositol-5-phosphatase (SHIP). Akt
activity can also be directly abolished through its dephosphorylation carried out by phosphatases like
the protein phosphatase 2A (PP2A) or the H domain and leucine-rich repeat protein phosphatase
(PHLPP) [12,13].
2. Viral Entry
In the course of the virus life cycle, viral attachment to the host cell is the earliest time point at
which host signaling pathways are activated. Interaction with the viral receptor embedded within the
host cell membrane activates and triggers a signaling cascade coincident with viral entry to set the
stage for a favorable cellular environment for viral needs. For example, within a minute after HIV-1
exposure, more than 200 phosphorylation sites are modified in T-cells, with the potential to alter
several cellular processes immediately after infection and thus to support viral replication [14].
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Figure 1. Schematic drawing of the PI3K/Akt signaling pathway. The phosphatidylinositol
3-kinase (PI3K) is activated through receptor-binding (receptor tyrosine kinases (RTKs) or
G protein-coupled receptors (GPCR), sidled blue line) by growth factors (GF) or cytokines
(CY) resulting in phosphorylation of PIP2. PIP3 subsequently serves as a second messenger
allowing the binding of pleckstrin homology domain-containing proteins like Akt. Thereby
the latter undergoes conformational changes leading to its phosphorylation and activation
by PDK1 and mTORC2. Akt finally participates in the regulation of cellular processes like
translation, cell growth and apoptosis by phosphorylating further proteins. Termination of
the signaling cascade can either occur through the dephosphorylation of PIP3 by the
phosphatase PTEN or SHIP, or further downstream through the dephosphorylation of Akt
by PHLPP or PP2A.
In general, enveloped viruses can enter their host cell via two routes (for review see [15]). Using a
pH-independent route, the virus attaches to its host cell and by interacting with its corresponding
host-cell receptor induces conformational changes of the viral glycoprotein causing the fusion of the
viral envelope with the plasma membrane of the target cell. This directly leads to the delivery of the
capsid into the cytoplasm followed by its uncoating. In a second route which is pH-dependent, the
virus enters the cell via endocytosis, a process essential in eukaryotes to internalize extracellular
molecules. In this process the virus is first engulfed into early endosomes, which later on mature into
late endosomes with a low pH, inducing the necessary conformational change of the viral glycoprotein
to induce fusion of the viral and endosomal membranes. Non-enveloped viruses can also enter through
endocytotic mechanisms, however they cross this internal membrane by pore formation or penetration
of the membrane [15] (Figure 2).
In immunological terms, endocytosis represents an inconspicuous way of entering a host cell by
delaying the immune recognition of infection but still guarantees a subsequent ferry to subcellular sites
of viral replication (for review see [16]). The success of this pathway may explain its use by a
multitude of viruses. Importantly, the attachment of the virus to the host cell membrane not only seems
to be a suitable instance for activating the PI3K/Akt signaling pathway for already triggering a desired
cellular surrounding; rather it seems that the pathway is directly involved in the entry of various
viruses by facilitating endocytotic uptake.
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Figure 2. Overview of viruses utilizing the PI3K/Akt signaling pathway during host cell
entry. (a) Viruses using the pH-dependent route to enter the host cell are engulfed in
endosomes and released inside the cell. Attachment of influenza A or vaccinia virus
(VACV) results in a clustering of lipid rafts at the cell surface (here: accentuated part of the
membrane), which is followed by PI3K/Akt-supported endocytosis. Inhibition of PI3K
(LY294002 or Wortmannin) reduces the infection by both viruses. The endosomal entry of
Hepatitis C virus (HCV), Avian leucosis virus (ALV), and African swine fever virus
(ASFV) is also diminished after inhibition of this signaling pathway. In the case of Zaire
Ebola virus (ZEBOV) the signaling pathway is important for further trafficking into the
cell as inhibition traps virus particles in vesicular compartments; (b) Likewise, the
pH-independent entry of herpes simplex virus 1 (HSV-1)—carried out through fusion of
the viral envelope with the plasma membrane—relies on PI3K activity. The receptor
binding of human immunodeficiency virus (HIV-1) already affects several hundred
intracellular phosphorylation events, which potentially support the viral life cycle. The
respective viral receptors are shown in green.
This is the case for the enveloped influenza A virus, whose binding to its sialic acid receptor results
in the clustering of plasma membrane lipid rafts [17] (Figure 2). These are small domains within the
plasma membrane enriched for cholesterols and glycosphingolipids [18]. As they contain different sets
of proteins—mainly signaling molecules—lipid rafts are thought to be involved in signal transduction
(for review see [19]). The clustering of such lipid rafts after influenza A attachment subsequently leads
to the activation of at least two RTKs followed by signaling events through the PI3K/Akt signaling
pathway, which promote viral internalization [20], supported by the cross-interaction of PI3K with the
Ras signaling pathway [21]. Moreover, acidification of the endosomal interior, necessary for fusion of
the viral and endosomal membranes is in part controlled by ERK and PI3K by upregulating the
vacuolar H+-ATPase activity. Based on immunoprecipitation experiments using extracts from the
epithelial cell lines MDCK and A549, direct interactions between the phosphorylated forms of
ERK 1/2 and the PI3K p85α subunit with the E subunit of this proton pump have been shown [22].
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Infection with the enveloped vaccinia virus (VACV) also causes a clustering of lipid rafts at the
host plasma membrane, leading to co-localization of virus and raft-associated integrin β, which finally
activates the PI3K/Akt signaling pathway to support endocytosis of the virus [23,24].
The enveloped Zaire Ebola virus (ZEBOV) likewise enters the host via the cellular endocytotic
pathway. In this case, the PI3K/Akt signaling pathway appears to be essential for a step immediately
after viral uptake, as inhibition of the cascade leaves virus particle stuck in vesicular compartments,
compatible with an arrest of further trafficking within the cell [25]. Recently an early and transient
activation of Akt by the enveloped hepatitis C virus (HCV) was also reported, which is most certainly
achieved through an interaction between the viral envelope protein E2 and its cellular co-receptors
CD81 and claudin-1. This early activation is essential for HCV entry, whereas later in infection Akt
activity seems dispensable [26].
Infection with the enveloped, acute-transforming oncogenic retrovirus, avian leucosis virus (ALV),
results in a transient, PI3K-dependent phosphorylation of Akt as early as 15 minutes post infection.
Moreover, treatment of cells with either PI3K inhibitor LY294002 or Wortmannin prior to infection
significantly reduces viral replication in a dose-dependent manner [27], pointing to a role of the
pathway during the entry mechanism.
The enveloped herpes simplex virus type-1 (HSV-1) attaches to the cell by heparan sulfates leading
to the fusion of the viral envelope with the plasma membrane. In addition to this, the infection is
accompanied by cytoskeletal changes facilitating filopodia formation and thus membrane fusion. The
filopodia formation leads to the activation of Rho-GTPase signaling [28]. The PI3K signaling pathway
seems to be involved in both, as inhibition prior to infection blocks Rho-GTPase signaling, reduces
filopodia formation and thus RhoA activity [29]. Activation of signaling events typical of
macropinocytosis also seems to be activated directly, following cellular entry of the highly pathogenic
African swine fever virus. This virus not only induces two critical effectors, Pak1 and Rac1, which link
Rho-GTPases to cytoskeleton reorganization and thus membrane perturbations and actin remodeling,
but also to the EGFR and PI3K/Akt pathway. Consequently, specific inhibitors of EGFR, i.e., 324674
or the phytoestrogen genistein, as well as inhibition of PI3K with LY294002, analyzed by the
phosphorylation status of its specific substrate PIP2, efficiently reduced viral uptake [30].
Although the molecular mechanisms within the PI3K pathway leading to endocytotic uptake still
remain elusive in many cases, the usage of this pathway for assisting viral uptake seems to be a
widespread viral strategy [20,21,23,25–27,29,30] (Figure 2).
3. Pre-mRNA Splicing
Eukaryotic pre-mRNA splicing is a process during which introns are removed and the flanking
exons ligated to form the mature mRNA. Alternative splicing enables differential usage of splice sites,
thus generating various transcript isoforms originating from one genetic template and increasing the
proteomic diversity (for recent review see [31]).
The usage of alternative splice sites is regulated by trans-acting splicing factors like SR (serine
arginine rich) proteins or hnRNPs (heterogeneous nuclear ribonucleoproteins) which bind to
cis-regulatory elements in the neighborhood of the splice sites (for review see [32]). Both classes of
these splicing regulators show dual and antagonistic functionality in a strict position-dependent
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manner [33]. Accordingly, expression, positioning and activity of these proteins are critical to splice
site selection. A growing body of evidence reveals the contribution of signaling pathways controlling
alternative splicing by transmitting incoming signals to the splicing machinery. This allows a faster
reaction to changing environmental conditions as transcript variants of different stability or protein
isoforms can be generated in the absence of a new round of transcription.
In response to extracellular stimuli, Akt indirectly acts on splicing regulation by phosphorylating
serine/threonine-protein kinase 2 (SRPK2) or by inducing the autophosphorylation of serine/threonine-protein
kinase 1 (SRPK1), leading to the translocation of both kinases into the nucleus thus modifying SR
protein activities [34,35]. Furthermore, SRSF1, SRSF7 and SRSF5 can directly be phosphorylated by
Akt itself [36–38] (Figure 3).
Figure 3. PI3K/Akt contribution to the regulation of alternative splicing and viruses
utilizing the pathway for splicing regulation. After PI3K-mediated phosphorylation, Akt
indirectly—through SRPK1/2—or directly phosphorylates SR proteins resulting in their
translocation into the nucleus where they bind to corresponding pre-mRNAs. hnRNP
proteins can also be targeted by Akt-mediated phosphorylation. Measles virus (MV)
interferes with PI3K activity, leading to altered SR protein phosphorylation and
subsequently to the alternative splicing of a constitutively active isoform of the PIP3
phosphatase SHIP, namely SIP110, which results in the down regulation of Akt activity
(this route is marked with blue arrows). The adenovirus E4-ORF1 protein and herpes
simplex virus 1 (HSV-1) control SR protein activity in support of viral replication either
through modulating PP2A or SRPK1 activity. Human immunodeficiency virus (HIV-1)
seems to depend on PI3K activity for accurate splicing of its own mRNA.
However not only SR proteins but also hnRNP L was demonstrated recently to be phosphorylated
in an Akt-dependent manner leading to the exclusion of 4 exons from the caspase-9 pre-mRNA.
Skipping of exons 3, 4, 5, and 6 results in the anti-apoptotic caspase-9b isoform, whose protein product
interferes with the formation of a functional Apaf-1-caspase-9 complex [39,40]. Inclusion of these four
exons, on the other hand, led to the synthesis of the antagonistic protein isoform of caspase-9, which is
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necessary for induction of apoptosis by the pro-apoptotic Bcl-2 family members [39], calling for a
precise regulation of these particular splice sites. The study elegantly refutes the dogma that the
PI3K/Akt pathway only modulates alternative splicing through the regulation of SR proteins.
Concerning apoptosis, Acinus—a component of the apoptosis-and splicing-associated protein
complex (ASAP) which is present in functional spliceosomes [41,42]—was also shown to be directly
phosphorylated by Akt, at least during chromatin condensation, leading to the inhibition of its
pro-apoptotic function [43]. This might be a clue to the general role of Akt in regulating the function of
the splicing relevant ASAP complex, once again emphasizing the close relationship between splicing
and apoptosis. These examples underline the significance of intermediated kinases, like PI3K and Akt,
in transforming incoming signals via the regulation of alternative splicing.
Viruses also profit from the presence of multiple cellular protein isoforms and apparently are able to
regulate their synthesis. In T-cells, for example, the single-stranded RNA measles virus blocks
PI3K/Akt activity, and thereby down regulates SR protein phosphorylation and their translocation into
the nucleus. As a result, a constitutively active splicing isoform of the lipid phosphatase SHIP, termed
SIP110, containing intronic sequences between exon 5 and 6, is expressed. This, in turn, leads to an
increase in PIP2 levels and consequently to a down regulation of activated PI3K levels, necessary for
activating T-cell proliferation [44]. In this particular case, the virus inhibits the signaling pathway
instead of stimulating it, emphasizing the ability of viruses to trigger cascades in either direction.
Recently, a global role of the PI3K signaling pathway in T-cell activation was revealed [45]. Using
exon arrays, Riedel et al. [45] showed that the inhibition of PI3K interferes with T-cell activation
through altered mRNA expression levels and alternative splicing patterns.
Expanding their coding potential, many viruses take advantage of the host splicing machinery to
ensure the production of their own protein diversity and to regulate the different stages of infection by
temporally expressing transcripts. Adenoviruses and HSV-1, both containing a double-stranded DNA
genome, seem to manipulate mRNA splicing in favor of viral gene expression and replication. Both
viruses do so via hypophosphorylation of particularly two SR proteins, SRSF1, SRSF9 (Adenovirus)
and SRSF3, SRSF5 (HSV-1), by modifying either PP2A or SRPK1activity [46,47] (Figure 3).
In monocyte-derived macrophages, the expression levels of hnRNP A1, hnRNP A2/B1 and hnRNP
H decrease upon human immunodeficiency virus 1 (HIV-1) infection, reaching levels comparable to
untreated control cells 2 weeks after infection. SRSF2 levels, on the other hand, are increased
2–3 weeks post infection and then decline again [48]. In CD4+ T-cells, the phosphorylation status of
six SR proteins—including SRSF2—and five other splicing related proteins immediately changed after
HIV-receptor interaction. Furthermore, most of these proteins were crucial for balanced HIV-1
transcripts and thus p24 levels [14].
Interestingly, the PI3K signaling pathway seems to be involved in HIV-1-mediated phosphorylation
of SR proteins, as inhibition of the kinase results in an altered phosphorylation pattern of some SR
proteins and concomitantly in an alternative splicing pattern of HIV-1 mRNAs, accompanied by a
significant reduction of viral replication [49]. These observations suggest an HIV-1-induced
PI3K/Akt-dependent modulation of splicing regulation, which seems to be essential for the viral life
cycle. In sum, these results emphasize the importance of splicing regulatory proteins for proper HIV-1
splicing, yet the impact on cellular alternative splicing and reprogramming in favor of viral replication
has not yet been examined. It will take further effort to uncover the number of viruses targeting the
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cellular splicing machinery to support and modulate gene expression. The identification of the cellular
players concerned will likewise help decipher the biological relevance of the PI3K/Akt pathway
regarding the regulation of pre-mRNA processing.
4. Cell Survival
Maintaining cell survival by acting downstream of growth factors is another key function of the Akt
kinase (for review see [50] and [51]). For this purpose Akt interferes with pro-apoptotic molecules to
inhibit their activity either through a direct phosphorylation of the regulators as in the case of several
Bcl-2 homology domain 3 (BH3)-only proteins (e.g., BAD), or indirectly through the phosphorylation
of certain transcription factors such as FOXO1 which thereupon translocate out of the nucleus and thus
can no longer promote the transcription of their pro-apoptotic target genes [52,53] (Figure 4).
As survival of the host cell ensures maintenance or replication of the intracellular viruses until the
infectious particles are assembled and spread throughout the organism, it is of no surprise that quite a
lot of work has been done to investigate virus-induced Akt-dependent anti-apoptotic mechanisms.
Figure 4. As a key regulator of cellular survival the PI3K/Akt signaling pathway is often
triggered by viruses. Upon incoming survival signals, the Akt-mediated inhibitory
phosphorylation of pro-apoptotic molecules such as BAD prevents the induction of the
caspase cascade. In addition, the inhibitory phosphorylation of the transcription factor
FOXO1 by Akt blocks its translocation into the nucleus and thus prevents the expression of
pro-apoptotic genes. A variety of viral proteins steps into this regulatory network to assure
cell survival, and thus maintaining the cellular environment for viral replication. Known
viral proteins interacting with the cellular member of the signaling pathways are
highlighted in red.
An HIV-1 Nef-mediated inhibition of PI3K activation through preventing the interaction of PI3K
with the platelet-derived growth factor receptor was demonstrated by Graziani and colleagues in
1996 [54]. Later a Nef-mediated activation of PI3K, which resulted in the inhibitory phosphorylation
of the pro-apoptotic factor BAD blocking premature apoptosis in T-cells, was observed. Although the
decline of T-cells is characteristic of progressive HIV infection, this early delay may ensure cellular
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survival until viral particles are assembled and released from the cell [55]. The authors suggest that
these conflicting observations could be the result of a different cellular localization of Nef during the
experiments. Graziani and colleagues used a stable cell line expressing Nef mostly in the cytoplasm
and not directly at the plasma membrane [54], at which Wolf and coworkers showed the protein to be
necessary for the activation of PI3K signaling [55]. Although ectopic expression of viral genes might
be a useful, and sometimes indispensable tool to unravel protein interactions, there is also an inherent
risk of missing out on the temporal and spatial aspects of kinase activation during viral replication.
Several groups have established that the influenza A non-structural protein 1 (NS1), known for its
role in suppressing host immune responses [56], directly activates the PI3K/Akt pathway to secure
anti-apoptotic signaling by interacting with the PI3K regulatory subunit p85 [57–60]. In contrast,
Jackson and coworkers suggested an NS1-mediated, but PI3K-independent, prevention of apoptosis as
NS1 mutant viruses induced apoptosis, while FCS-activated Akt was not able to overcome this
effect [61]. Even though strain-specific viral requirements for NS1-activated PI3K have been
discovered, mere strain-specificity cannot explain the observed differences concerning the role of PI3K
in NS1-mediated anti-apoptotic signaling, since both labs used the same influenza A strain [62,63].
However, Ayllon and colleagues, using mouse-adapted viruses, demonstrated in vivo subtle differences
in NS1 protein localizations depending on the NS1 isoform, which only differs in seven amino acids
between the two strains, A/Puerto Rico/8/34 (PR8) and A/WSN/33. Applying a cell-based assay, they
observed that PR8/NS1-induced PIP3, the target of PI3K, appeared to accumulate in microdomains
whereas WSN/NS1-induced PIP3 was broadly distributed throughout the plasma membrane, which
may explain that different NS1 variants define intracellular sites of PI3K activation [62,63]. To
entirely decode how influenza A virus impacts the PI3K signaling pathway, the authors suggest
carefully selecting the viral strain, host-cell type, time post-infection and the PI3K isotype to be used
in further experiments [62].
HSV-1, with a genome of approximately 150 kb encoding more than 70 proteins, is able to afford
its own serine/threonine kinase. Nonetheless, early during HSV-1 infection, apoptosis is still blocked
in an Akt-dependent manner until the virus has accumulated enough Us3 protein kinase to mimic Akt
activity. However, in the absence of Us3, the virus appears to have evolved a backup mechanism to
retain the activity of Akt thereby ensuring anti-apoptotic signaling [64]. Another herpes virus thought
to manipulate the PI3K/Akt-signaling pathway is the human cytomegalovirus (HCMV). Both the
HCMV major immediate-early proteins (MIEPs) and a constitutive active form of Akt can inhibit
temperature-induced apoptosis in ts13 cells. Since this ability of the MIEPs to inhibit apoptosis is lost
when PI3K/Akt signaling is inhibited by LY294002 and since MIEPs can activate Akt, it was
concluded that the MIEPs induced anti-apoptotic activity is Akt-mediated [65].
A third member of the herpes virus family, Epstein-Barr virus (EBV), induces the PI3K/Akt
pathway through the viral latent membrane protein 1 (LMP1), resulting in host-cell survival,
which most probably contributes to EBV persistence in B cells necessitating sustained apoptotic
inhibition [66]. Both viral EBV transcriptional activators, BZLF1 and BRLF1, can reactivate the lytic
form of viral replication with BRLF1 acting in a PI3K/Akt-dependent manner as inhibition of PI3K
abolishes BRLF1-induced transcriptional activation [67]. Recently, however, for two other herpes
viruses, murine gamma herpesvirus-68 (MHV-68) and human herpesvirus-8/Kaposi’s sarcoma-associated
herpesvirus (HHV8/KSHV), Peng and coworkers demonstrated that Akt promotes viral persistence by
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suppressing transcriptional reactivation of these viruses rather than reactivating lytic replication. In this
case reactivation or the transition from latency to lytic replication is controlled by the viral
transcription activator (RTA), an immediately-early (IE) gene, whose activity is negatively regulated
by the PI3K/Akt pathway [68]. They observed enhanced MHV-68 production in permissive fibroblast
after either LY294002 treatment in a dose-dependent manner or RNAi-mediated Akt1 silencing. For
the various other capabilities of the virus to modulate host cell pathways for longer periods the reader
is referred to the detailed review by Cooray [69].
Two members of the poxvirus family, VACV—which already uses the pathway to assist its
endocytotic uptake [23]—and cowpox virus (CPXV), also hijack the PI3K/Akt signaling pathway to
prevent apoptosis. Inhibition of Akt activity either by the pharmaceutical PI3K inhibitor LY294002 or
by expressing a dominant-negative form of Akt reduces viral titers by up to 90%, corresponding to
cleavage of caspase-3 and PARP as significant indicators of apoptotic cells [70].
In rotavirus infected cells an increased Akt phosphorylation depends on a direct interaction between
the viral nonstructural protein NSP1 and PI3K. This interaction leads to the Akt-dependent inactivation
of pro-apoptotic proteins on the one hand and the activation of NFkβ-dependent induction of
anti-apoptotic genes on the other [71,72]. An association of these two survival pathways has been
known for a long time [73] and Bagchi and coworkers suggest these to be partially overlapping or even
cooperative in case of rotavirus infection [71]. Dengue virus and japanese encephalitis virus, both
members of the single-stranded RNA flavivirus family, also make use of a PI3K-dependent blocking
of apoptosis as early as 15 min after infection, indicating that the mere viral attachment leads to an
activation of the signaling pathway [74]. However, treatment of infected cells with the PI3K inhibitors
LY294002 or Wortmannin did not alter effective viral replication. Nonetheless the PI3K/Akt signaling
appears to protect the infected cells from early apoptosis and thereby offers more time to generate
virus progeny.
Besides its beneficial role during HCV entry [26], the PI3K/Akt pathway also mediates survival
of the HCV-infected cells [75,76]. The replication of HCV is accompanied by the formation of lipid
rafts, followed by an increased production of the proto-oncogene N-Ras, a known activator of the
PI3K-signaling pathway [77]. This consequently results in Akt activity leading to protection against
apoptosis. Interestingly, upon inhibition of either signaling molecules, the levels of HCV replication is
raised. As this virus establishes chronic infection in most of the cases, Mannova and Beretta claimed
that the activation of the Ras-PI3K-Akt signaling pathway may ensure both cellular survival and the
maintenance of persistent infection by maintaining the virus production at a low level [76].
As respiratory syncytial virus (RSV), coxsackievirus B3 (CVB3), avian reovirus (ARV), rubella
virus and SARS coronavirus also utilize the PI3K-mediated cell survival [78–82], this highlights the
obvious advantage viruses enjoy from the prolonged cell survival.
5. Viral Translation
Protein synthesis requires many different enzymes, none of which are encoded by viral genomes.
As viruses use the cellular translation machinery for protein synthesis, their mRNAs must efficiently
be recognized by the cellular ribosomal 40S subunit, either in a cap-dependent or cap-independent manner.
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One indirect downstream target of Akt is the kinase mTOR (mammalian target of rapamycin)
within the mTOR complex 1 (for review see [83]). This kinase is necessary for the regulation of
cap-dependent translation carried out by the initiation complex eIF4F. After its inhibitory
phosphorylation by activated Akt kinase [84], the TSC is unable to stimulate the intrinsic GTPase
activity of Rheb, which finally leads to the maintenance of high levels of GTP. GTP then removes
FKBP38 from mTORC1, thus restoring the latters’ activity. Active mTOR in turn phosphorylates both
the p70S6 kinase (S6K), which promotes translation elongation and ribosome biogenesis, and the
eIF4E binding protein (4E-BP). Phosphorylated 4E-BP cannot prevent formation of the eIF4F
complex—consisting of the cap-binding subunit eIF4E, the RNA helicase eIF4A and the scaffolding
protein eIF4G—thus translation can be initiated by the recruitment of the preinitiation-complex to the
7-methylguanosine (m7G) cap of the 5' end of the mRNA [85] (Figure 5). Hence viruses, depending on
cellular 5’cap-dependent translation, must establish a strategy to either maintain mTOR activity or
directly ensure eIF4F complex formation.
Figure 5. PI3K/Akt-mediated mTOR activation positively regulates protein synthesis.
Inhibitory phosphorylation by Akt prevents TSC-mediated stimulation of the GTPase
Rheb, leading to the activation of mTOR. Activated mTOR then phosphorylates 4E-BP,
thereby enabling eIF4F cap-binding complex formation and thus cap-dependent translation.
It also phosphorylates S6K to facilitate ribosome biogenesis. Human cytomegalovirus
(HCMV)-mediated phosphorylation of the eIF4F subunit eIF4E is both mTOR- and
PI3K-independent, whereas its phosphorylation of 4E-BP is dependent on PI3K. This is
also the case for the vaccinia virus (VACV)-mediated 4E-BP phosphorylation. Epstein-Barr
virus (EBV) and adenovirus on the other hand seem to regulate translation in a PI3K and
mTOR-dependent manner. Known viral proteins interacting with the cellular member of
the signaling pathways are highlighted in red.
HCMV-induced Akt phosphorylation seems to regulate, in addition to the prolonged cell survival [65],
viral translation. Treatment of infected cells with the pharmaceutical PI3K inhibitor LY294002
reduces viral titers by more than 4 logs. Surprisingly, rapamycin (an mTOR inhibitor), as opposed to
LY294002 treatment, does not diminish viral protein synthesis, indicating an HCMV-induced,
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mTOR-independent mechanism to maintain translation [86]. Later it was indeed found that HCMV
phosphorylates two downstream targets of mTOR: the transcriptional repressor 4E-BP and the
scaffolding protein eIF4G to maintain the activity of the eIF4F complex. Thereby, both phosphorylations
are in fact mTOR-independent, whereas the phosphorylation of 4E-BP is PI3K-dependent, explaining
the diminished viral protein synthesis after PI3K inhibition [87].
Additionally, HCMV enhances the activity of the cap-binding unit eIF4E [88] and also activates
rictor, the kinase of Akt S473 [89]. Remarkably, both the raptor complex (mTORC1) and the rictor
complex (mTORC2) can mediate an inhibitory phosphorylation of 4E-BP and a stimulatory
phosphorylation of p70S6K. This indicates that upon HCMV infection the substrate specificity of
rictor and raptor is modified, correlating with a beneficial rictor-mediated Akt activation.
Yet another member of the herpes virus family, EBV, affects, in addition to the regulation of
apoptosis [66], the cellular translational machinery. The viral protein LMP2A activates mTOR1, most
certainly via the PI3K/Akt signaling pathway since the activation is sensitive to the PI3K inhibitor
Wortmannin. This leads to an enhanced disassociation of 4E-BP from eIF4E and thus to an activation
of the cap-dependent translation [90].
VACV gains, besides the facilitated viral uptake and the prevention of apoptosis [23,70], a third
benefit out of the PI3K/Akt signaling activation. The increased phosphorylation level of Akt post
infection corresponds with a decreased level of repressor 4E-BP bound to eIF4E. Inhibition of the
PI3K, but not the mTOR1 pathway, suppresses the production of VACV proteins, indicating an
mTOR-independent but PI3K-dependent mechanism to regulate 4E-BP repressor activity, as in the
case of HCMV [91]. Two of the human papillomavirus (HPV) early proteins, E6 and E7, not only
activate Akt/mTOR signaling but also maintain the phosphorylation status by inhibiting the Akt
phosphatase PP2A. This results in an inactivation of 4E-BP and in an activation of S6K, supporting
viral cap-dependent protein synthesis [92,93]. These signaling events were recently also shown to be
triggered by the HPV-mediated EGFR stimulation during viral entry [94].
Besides the modification of SR protein phosphorylation [46], adenoviruses also activate the
mTOR1 pathway [95]. Two viral proteins, E4-ORF1 and E4-ORF4, function to mimic incoming
signals to activate this pathway. Interestingly, the proteins seem to act via different mechanisms as
only E4-ORF1 is necessary and sufficient for the PI3K-dependent activation of S6K, whereas
E4-ORF4 acts independently of PI3K. Both proteins seem to collaborate to induce mTOR activity,
independent of the stimulation by nutrients or growth factors.
Taken together, the manipulation of cap-dependent protein synthesis—irrespective of the
involvement of mTOR1—is a common mechanism of viruses to guarantee an efficient translation of
viral proteins.
6. Conclusions
In the past decades our knowledge of virus-induced modifications of host signaling pathways has
grown rapidly and the diversity of viruses interfering with the PI3K/Akt pathway notably highlights
the importance of this research field. Table 1 summarizes interacting viral proteins and Figure 6
additionally points out the remarkable temporal range of processes during viral life cycles affected by
either transient or long-term activation of the PI3K pathway.
Viruses 2013, 5
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Table 1. Viruses and viral proteins (if known) that interact with the PI3K/Akt signaling
pathway during different steps of the viral life cycle.
Viral Life Cycle
Entry
pre-mRNA
Splicing
Cell Survival
Translation
Virus
Protein
Function
Reference
Influenza
VACV
HCV
E2
Virus internalization;Endosomal acidification
Integrin β1-dependent virus entry
Facilitation of virus entry
[20,22]
[23]
[26]
ALV
HSV-1
ASFV
HIV-1
-
[27]
[29]
[30]
[49]
Adenovirus
E4-ORF4
Facilitation of virus entry
Filopodia formation; Fusion
Macropinocytosis
Alternative splicing of viral mRNAs
Dephosphorylation of SF2/ASF and SRp30c;
[46]
MV
HIV-1
Influenza
Nef
NS1
splicing of viral mRNAs
T-cell proliferation
Anti-apoptotic effects
Anti-apoptotic effects
VACV
HCV
CPXV
DENV
JEV
NS5A
-
Anti-apoptotic effects
Anti-apoptotic effects; viral persistence
Anti-apoptotic effects
Anti-apoptotic effects
Anti-apoptotic effects
[70]
[75,76]
[70]
[74]
[74]
HCMV
Rotavirus
EBV
RSV
CVB3
MIEPs
NSP1
LMP1
-
Anti-apoptotic effects
Anti-apoptotic effects
Persistence
Anti-apoptotic effects
Anti-apoptotic effects
[65]
[71]
[66]
[78]
[79]
ARV
VACV
HCMV
Adenovirus
EBV
E4-ORF1
LMP2A
Anti-apoptotic effects
Translation initiation via activity of 4E-BP
Translation initiation via activity of 4E-BP
mTOR activation
mTOR activation
[80]
[91]
[87]
[95]
[90]
HPV
E6, E7
mTOR activation
[93]
[44]
[54,55]
[57–59]
In general, unraveling of signaling pathways is a challenging task, especially where viral
interference occurs. The sensitive regulatory system can be easily disturbed by experimental conditions
used to mimic the infection of a complex organism. In particular the cell lines used may omit the
complex network of cross-talk between signaling pathways in a real infection situation. Moreover,
many cancer T-cell lines, which are commonly utilized when concerning infections, often carry
mutations in genes encoding members of the PI3K/Akt signaling pathway [96,97]. This is of no
surprise bearing in mind the role of the pathway in the regulation of apoptosis and proliferation.
Nevertheless, the growing body of evidence of the continuous interaction between the viral survival
strategies and the host cell-induced defense mechanisms, such as apoptosis, not only helps us
understand the viral strategy of taking over control of cellular processes, but also offers additional
insights into general mechanisms of such processes. New methods like next generation sequencing and
high throughput protein identification are promising tools to track the viral-host interactions and to
Viruses 2013, 5
3205
monitor the impact on several levels of the host cell metabolism, finally supporting the development of
antiviral therapies.
Figure 6. Time schedule of viral-induced interference with PI3K/Akt signaling based on
the time point of activation and benefit during viral life cycles. Early activation of the
PI3K/Akt signaling pathway during the first contact with the cell or a few minutes after
entry can lead to both short-term cellular answers to facilitate viral entry and long-term
reactions (lasting for hours or even days) to support viral and cellular splicing, viral
translation and cell survival. Later activation, hours or days post-infection, can likewise
promote such long-term effects.
Acknowledgments
We are grateful to Colin R. Mackenzie for critical reading and editing of the manuscript. We thank
Frank Hillebrand and Marek Widera for helpful comments. N.D. is funded by a Jürgen Manchot
foundation fellowship.
Conflicts of Interest
The authors declare no conflict of interest.
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